Not Applicable.
The present invention relates to a method and apparatus for cryopreservation of biological material using ultra rapid freezing.
With recent advances in cell transplantation, tissue engineering and genetic technologies, the living cell is becoming an important therapeutic tool in clinical medical care. From the use of living artificial skin and bone material to treat burn and trauma victims, to bioartificial devices and direct transplantation of cellular material to treat the increasingly long list of genetically-based diseases, living cells are increasingly incorporated into comprehensive treatment. In such a construct, the exogenous cells perform the multitude of complex tasks which the diseased tissue cannot. Successful long-term preservation and storage of mammalian cells is critical to the success of this type of medical care.
Conventional cryopreservation protocols rely on the addition of cryoprotectants to control the formation of damaging crystalline ice in the intracellular and extracellular liquid. The formation of ice in the extracellular liquid leads to dehydration of cells and has also been shown to catalyze the formation of intracellular ice [Toner et al., xe2x80x9cThermodynamics and kinetics of intracellular ice formation during freezing of biological cells,xe2x80x9d 67 J. Applied Phys. 1582-1593 (1990)]. The formation of intracellular ice directly damages the cell and usually leads to cell death.
Equilibrium and non-equilibrium cryopreservation protocols try to balance the deleterious effects of cell dehydration, exposure of the cells to toxic cryoprotectants and the lethal formation of intracellular ice so as to yield the highest possible percentage of viable cells [Mazur, xe2x80x9cEquilibrium, Quasi-equilibrium, and non-equilibrium freezing of mammalian embryos,xe2x80x9d 17 Cell Biophysics 53-92 (1985)]. Protocols of this type have been very successful for certain cell types. For example, survival rates of greater than 90% have been reported for erythrocytes [Nei, xe2x80x9cFreezing injury to erythrocytes I. Freezing patterns and post-thaw hemolysis,xe2x80x9d 13 Cryobiology 278-286 (1976)], pancreatic islets [Jutte et al., xe2x80x9cVitrification of Human Islets of Langerhans,xe2x80x9d 24 Cryobiology 403-411 (1987)] and mouse oocytes [Karlsson et al, xe2x80x9cFertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol,xe2x80x9d 11 Human Reproduction 1296-1305 (1996)].
There are many cell types, however, for which acceptable cryopreservation protocols have not been developed. This is largely due to the fact that the concentration of cryoprotectant required to avoid intracellular ice formation is too high for most cells to tolerate [Fahy et al, xe2x80x9cVitrification as an approach to cryopreservation,xe2x80x9d 21 Cryobiology 407-426 (1984)]. Among the important cell types for which successful and reliable freezing protocols have not been developed are hepatocytes, human oocytes, platelets and granulocytes. The fact that human oocytes have not been preserved successfully in spite of the successful freezing of mouse oocytes illustrates another difficulty with the current methods of cryopreservation: they are extremely dependent on cell type. Even closely related cell types behave and survive differently when cryopreserved.
An alternative to conventional approaches to cryopreservation by freezing with high levels of cryoprotectant is vitrification, i.e., solidification of a liquid into an amorphous or glassy state as opposed to the crystalline state. Unlike the liquid-to-crystal transition, the liquid-to-glass transition is generally believed not to have any adverse biological effects. This is because there is no elevation in electrolyte concentration, no ice crystals to cause mechanical damage, and no potentially damaging osmotic shifts during the vitrification of cell suspensions.
It appears that nearly all liquids would undergo a transition to a glassy state if crystallization is bypassed on cooling. A necessary and sufficient condition for this transition is that the liquid solution should be rapidly cooled to the glass transition temperature so that nucleation and crystal growth cannot occur. Typically, the requisite cooling rates are very high for water (approximately 107xc2x0 C./min), but they can be reduced to more workable levels (approximately 10xc2x0 C./min) by the addition of cryoprotectants (CPA, usually 50 to 60% w/w). However, CPA concentrations this high are typically lethal to biological cells. New methods of ultra-rapid cooling are needed to achieve glassy state during cooling of biological cell suspensions.
The formation of intracellular ice during freezing may be avoided by hyperquenching the cells. In hyperquenching the water is cooled so quickly that nucleation events do not occur and the liquid undergoes a glass phase transition. As liquid water is cooled below its freezing point, it becomes energetically favorble for nucleation to occur. At 130 K, however, liquid water goes through a glass phase transition, which is a second order thermodynamic phase transition, and the relaxation time for the molecules becomes greater than laboratory time scalesxe2x80x94i.e., the viscosity of the fluid increases so that molecular rearrangement into crystals becomes impossible. If one can get water to the glass phase before ice crystals nucleate, then one creates an amorphous solid referred to as either amorphous solid water or amorphous ice.
There are a number of ways to form glass phase solid water; water vapor deposition on to cryo-cooled plates at very low pressures, exposing crystalline ice to very high pressures at temperatures below 130 K and thereby crushing it to the glass phase, and spraying water micro-droplets at supersonic velocities onto cryoplates. None of these techniques, however, is suitable for use in freezing cells. Water vapor deposition simply cannot be done with a cell, and the other two methods expose the cell to lethal stress.
In order to preserve the wide variety of cellular material needed in current medical procedures, a new method of preservation that can successfully cryopreserve biological material without the formation of lethal intracellular ice is needed. Such a method must avoid the use of high concentrations of CPAs which are toxic to many cell types and not expose the biological material to damaging physical stresses.
The technique of the invention uses spatially confined heating to reduce or eliminate the effects of intracellular ice on cryopreserved biological material.
One method of the invention includes placing a cellular material sample in thermal contact with a cryogenically coolable environment. The cryogenically coolable environment achieves and is maintained at a temperature below the glass phase transition temperature of the cellular material for a time sufficient to cool the sample below its glass phase transition temperature. Spatially continued heating using radiant energy is then applied to at least a portion of the sample in order to cause thawing in at least a portion of the sample. The spatially confined heating is then stopped resulting in the cooling of the cellular material. This method results in cooling rates greater than 105xc2x0 C. per second, and even cooling rates greater than 106xc2x0 C. per second, resulting in the vitrification of the cellular material.
Alternatively, the cellular material sample may be provided in thermal contact with a cryogenically coolable environment that is maintained at a temperature below the glass phase transition temperature of the cellular material and subject to spatially confined heating using radiant energy so that at least a portion of the cellular material is melted. The cellular material so provided may be previously frozen and stored in a frozen state before being so provided, or it can be frozen by its contact with the cryogenically coolable environment. After the cellular material is so provided, the spatially confined heating is stopped, resulting in the rapid cooling and vitrification of the cellular material.
In specific embodiments, the spatially confined heating may be provided using a radiant energy source that is at least partially absorbed by the cellular material or by the water found within the cellular material. The cryogenically coolable environment may be provided by a cryostage or, in one embodiment, by suspending the cellular material within a hole formed in a high thermal conductivity material.
Material cryopreserved using the method of the invention may be recovered by warming the material at a warming rate sufficiently high so as to prevent devitrification, i.e. the nucleation of ice crystals from the glass phase during thawing.
In a further method of the invention, biological material is either cooled to a temperature below the glass phase transition temperature of the material by placing it in contact with a cryogenically coolable environment, or providing biological material at a temperature below its glass phase transition temperature. Radiant energy is applied to at least a portion of the biological material, that portion being unfrozen.